Work in our laboratory has led to the identification, isolation and characterization of several brain and pituitary endopeptidases involved in the metabolsim of peptide hormones and neuropeptides. Based on the mechanism of action and specificity of these enzymes we have designed and synthesized potent active site directed inhibitors that can be used as probes in studies on the role these enzymes in the metabolism of bioactive peptides. Recent work in our laboratory has laboratory has focused on two zinc-metalloendopeptidase, classified as Ec 3,4,24,15, and EC 3,4,24,11 ("enkephalinase"). While endopeptidase-24.11 appears to function primarily as a degradative enzyme, there are indications that endopeptidase-24.15 which occurs in both soluble and membrane-bound form, is involved in processing and metabolism of neuropeptides and peptide hormones. This enzyme, discovered in our laboratory, is highly concentrated in brain and pituitary. It generates enkephalins from precursors and is apparently involved in the metabolism of LHRH and neurotensis. The objective of this proposal is to extend our studies on the biochemistry and function of these enzymes and to define their role in the metabolism of these neuropeptides. Both forms of endopeptidase-24.15 will be isolated as homogeneous proteins and compared with respect to their physicochemical properties and specificity. Polyclonal and monoclonal antibodies will be raised against the two enzyme forms and used to isolate the enzymes by immunoadsorption chromatography, to compare their immunological properties, to study their localization by immunocytochemical methods, and to compare their immunological properties, to study their localization by immunocytochemical methods, and to compare their distribution with that of LHRH and opioid peptides. Potent, active site directed inhibitors of the enzymes, recently synthesized in our laboratory, will be used in experiments with brain slices to study the effect of enzyme inhibition on endogenous levels of these peptides, and on the extent of their recovery from depolarized brain slices. The same inhibitors will be administered by intracerebro-ventricular injection, in order to explore the effect of enzyme inhibition on endogenous levels of LHRH and opioid peptides. We will isolate and sequence cDNA(s) coding for endopeptidase-24.15 using an expression library prepared against mRNA from rat brain and AtT20 cells, then screen a genomic library and isolate and sequence the genes coding for the enzyme, and determine the anatomical sites of enzyme synthesis using nuclease protection and/or Northern blot analysis combined with in situ hybridization. The effect of castration, estrogens and androgens on the expression of both endopeptidase-24.15, and endopeptidase-24,11 will be explored in detail, since recent studies have shown that the activity of the two enzymes changes significantly in the pituitary, preoptic area and median eminence after castration and estrogen treatment, indicating that the activity of the two enzymes in under hormonal control at these sites.